NextSeq 550 paired end sequencing; CUT&RUN Negative Controls in Human and Mouse
CUT&RUN Negative Controls in Human and Mouse
HEK293T cells were harvested using TrypLE (Cat. #12604013, Gibco). Tissues were dissociated to single-cell via incubation in Tryple Express Enzyme (12-604-013, Thermo Fisher Scientific; 1 ml /4 limbs) for 15 minutes at 37C °C on a shaker. The cell suspension was resuspended in ice-cold PBS, filtered through a 40 m cell strainer (KKE3.1, Carl Roth) and further processed for CUT&RUN. Human embryonic kidney 293T cells (HEK293T) were cultured at 37 °C in a humidified incubator with 5% CO2. Cells were cultured in high glucose Dulbecco's Modified Eagle Medium (Cat. #41965039, Gibco) supplemented with 10 % bovine calf serum (Cat. #1233C, Sigma-Aldrich) and 1X Penicillin-Streptomycin (Cat. #15140148, Gibco). HEK293T cells were cultured in media containing 10 µM CHIR99021 (Cat. #SML1046, Sigma Aldrich), LGK, or no stimulation. Samples were washed twice in wash buffer (HEPES pH 7.5 [20 mM], NaCl [150 mM], Spermidine [0.5 mM], Roche Complete Protease Inhibitor EDTA-Free (Cat. # COEDTAFRO, Roche)) and bound to 20 µl Magnetic ConA Agarose beads (Cat. #ABIN6952467, antibodies-online) equilibrated in binding buffer (HEPES pH 7.5 [20 mM], KCl [10 mM], CaCl2 [1 mM], MnCl2 [1 mM]). Antibody incubation was performed in 150 µl antibody buffer (wash buffer with digitonin [0.01%] and EDTA [2 mM]) with 1.5 µl anti-β-catenin antibody (Cat. #ABIN2855042, antibodies-online) overnight at 4 °C. Thereafter washes were performed with wash buffer containing 0.01% digitonin. After overnight incubation, samples were washed three times and pAG-MN protein was added at 0.6 µg/ml, in total volume of 150 µl. pAG/MNase was a gift from Steven Henikoff (Addgene plasmid #123461; http://n2t.net/addgene:123461; RRID: Addgene_123461), expressed and purified as described in (REF Meers 2019a). Samples were washed three times, followed by digestion for 30 min in wet ice in wash buffer supplemented with 2 mM CaCl2. 2X stop buffer (NaCl [340 mM], EDTA [20 mM], EGTA [4 mM], digitonin [0.05%], RNase A [100 µg/ml], glycogen [50 µl/ml]) was added to stop the reaction, and samples were incubated for 30 min at 37 °C. Beads were collected on the magnet and liquid transferred to a new tube. 2.5 µl proteinase K (Cat. #P8107S, New England BioLabs) and 2 µl 10% SDS were added, and samples incubated for 1 hr at 50 °C. Cells were washed three times in Nuclear Extraction (NE) buffer (HEPES-KOH pH-8.2 [20 mM], KCl [10 mM], Spermidine [0.5 mM], IGEPAL [0.05%], Glycerol [20%], Roche Complete Protease Inhibitor EDTA-Free), resuspended in 40 µl NE per sample and bound to 20 µl Magnetic ConA Agarose beads equilibrated in binding buffer. After incubation nuclei and beads are resuspended for 5 min in EDTA wash buffer (wash buffer with EDTA [0.2 mM]). Samples were divided in 200 µl PCR tubes and antibody incubation was performed in 200 µl wash buffer with 2 µl of antibody (for antibody and batch information, see Table 1) ON at 4 °C on a rotator. After ON incubation samples were washed 5 times in wash buffer and resuspended in 200 µl of pAG-MN buffer (wash buffer with pAG-MN 0.6 µg/ml) for 30 min at 4 °C on a rotator. Samples were washed five times, followed by digestion for 30 min in wet ice in wash buffer with 2 mM CaCl2. After 30 min the digestion buffer was removed and the reaction was stopped with 50 µl of 1X Urea STOP buffer (NaCl [100 mM], EDTA [2 mM], EGTA [2 mM], IGEPAL [0.5%], Urea [8.8 M]) and the samples were incubated 1 hr at 4°C. Beads were collected on the magnet and liquid transferred to a new PCR tube in which it was cleaned up twice using Mag-Bind TotalPure NGS beads (Cat. #M1327, Omega Bio-Tek) at 2X, and then resuspended in 20 µl Tris-HCl pH 7.5. Library preparation was performed using the KAPA Hyper Prep Kit for Illumina platforms (Cat. #KK8504, KAPA Biosystems) according to the manufacturer's guidelines with the following modifications. End repair and A-tailing was performed with 0.4X volume reactions with 20 µl of purified DNA. The thermocycler conditions were set to 12 °C for 15 min, 37 °C for 15 min and 58 °C for 25 min to prevent thermal degradation of the shortest fragments. Adapter ligation was performed in 0.4X volume reactions. KAPA Dual Indexed adapters were used at 0.15 µM. A post-ligation clean-up was performed with Mag-Bind TotalPure NGS beads at 1.2X the sample volume. Resuspension was done in 10 mM Tris-HCl pH 8.0. Library amplification was performed in 0.5X volume reactions. The cycling conditions were set as follows: 45 sec initial denaturation at 98 °C, 15 sec denaturation at 98 °C, 10 sec annealing/elongation at 60 °C, 1 min final extension at 72 °C, hold at 4 °C, with 13 cycles. After amplification, a post-amplification cleanup was performed using NGS beads at 1.2X sample volume. Libraries were then run on an E-Gel EX 2% agarose gel (Cat. # G402022, Invitrogen) for 10 min using the E-Gel Power Snap Electrophoresis System (Invitrogen). Bands of interest between 150 and 500 bp were excised and purified using the GeneJET Gel Extraction Kit (Cat. #K0691, Thermo Scientific) according to manufacturer's instructions.